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Title
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X-ray crystallographic studies of Escherichia coli branching enzyme in complex with maltooctaose and rice branching enzyme I in complex with dodecaose
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Creator
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Fawaz, Remie
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Date
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2016
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Collection
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Electronic Theses & Dissertations
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Description
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ABSTRACTX-RAY CRYSTALLOGRAPHIC STUDIES OF ESCHERICHIA COLI BRANCHING ENZYME IN COMPLEX WITH MALTOOCTAOSE AND RICE BRANCHING ENZYME I IN COMPLEX WITH DODECAOSEByRemie FawazBranching enzyme plays a key role in determining the final structure of glycogen or starch; this outcome structure is unique to every species, therefore the diversity of branching enzymes structures. While the biosynthesis of the polymers constitutes of three major steps, the last reaction of the pathway catalyzed by...
Show moreABSTRACTX-RAY CRYSTALLOGRAPHIC STUDIES OF ESCHERICHIA COLI BRANCHING ENZYME IN COMPLEX WITH MALTOOCTAOSE AND RICE BRANCHING ENZYME I IN COMPLEX WITH DODECAOSEByRemie FawazBranching enzyme plays a key role in determining the final structure of glycogen or starch; this outcome structure is unique to every species, therefore the diversity of branching enzymes structures. While the biosynthesis of the polymers constitutes of three major steps, the last reaction of the pathway catalyzed by branching enzyme embodies the cleavage of the α-1,4 glycosidic bond and the transfer of the produced oligosaccharide to the specific α-1,6 position creating an α-1,6 branch point. This process results in highly branched polymeric structures, which represent major carbon sources and carbohydrate storage compounds in living organisms, and are essential both in nature and industry. The structure of Escherichia coli glycogen branching enzyme has been solved both in the apo and holo forms. Binding to linear and cyclic oligosaccharides has been studied and showed seven external binding sites, but binding in the active site was not seen. Oligomers longer than previously used were investigated in an attempt to see binding in the catalytic center, only to discover more peripheral binding sites seemingly independent of each other. Examining the binding sites’ locations and sugars’ orientations indicates that these sites probably cooperate together in order to hold the long sugar polymer on the surface of the protein during the branching reaction. Hypotheses regarding the mode of binding between E. coli branching enzyme and the sugar during the reaction, and the roles for key binding sites and residues in the mechanism are proposed.Rice branching enzyme I, a starch branching enzyme, was also previously crystallized and the structure of the truncated version was solved. Binding attempts on this protein showed surface binding sites as well. In an effort to better understand the mechanism of action of this enzyme, we crystallized the protein and soaked it in a 12-unit oligomer, which bound to the enzyme hanging over the active site without reaching into the groove. Two new binding sites were discovered for this protein and the residues involved were identified. There are several hydrogen bonds and aromatic stacking interactions within the binding sties. Hypotheses for the binding mode and reaction mechanism are proposed.In addition, we have worked on ADP-glucose pyrophosphorylases that catalyze the first step in the biosynthesis pathway. The small subunit of the protein expresses as a homotetramer that is highly active. Although the potato tuber form was previously expressed in a collaborating laboratory at extremely small levels, and the protein was crystallized in the inactive form in the Geiger lab, our intensive attempts only succeeded in purifying the protein of interest, but the enzyme would precipitate instead of concentrating.
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Title
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The effects of genetic background on the evolution of antibiotic resistance and its fitness costs
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Creator
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Card, Kyle Joseph
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Date
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2020
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Collection
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Electronic Theses & Dissertations
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Description
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Antibiotic resistance is a growing public-health concern. Efforts to control the emergence and spread of resistance would benefit from an improved ability to forecast when and how it will evolve. To predict the evolution of resistance with accuracy, we must understand and integrate information about many factors, including a bacterium's evolutionary history. This dissertation centers on the effects of genetic background on the evolution of phenotypic resistance, its genetic basis, and its...
Show moreAntibiotic resistance is a growing public-health concern. Efforts to control the emergence and spread of resistance would benefit from an improved ability to forecast when and how it will evolve. To predict the evolution of resistance with accuracy, we must understand and integrate information about many factors, including a bacterium's evolutionary history. This dissertation centers on the effects of genetic background on the evolution of phenotypic resistance, its genetic basis, and its fitness costs. To address these issues, I used Escherichia coli strains from the long-term evolution experiment (LTEE) that independently evolved for multiple decades in an environment without antibiotics.First, I examined how readily these LTEE strains could overcome prior losses of intrinsic resistance through subsequent evolution when challenged with antibiotics. Second, I investigated whether lineages founded from different genotypes take parallel or divergent mutational paths to achieve increased resistance. Third, I tested whether fitness costs of resistance mutations are constant across different genetic backgrounds. In these studies, I focused attention on the interplay between repeatability and contingency in the evolutionary process. My findings demonstrate that genetic background can influence both the phenotypic and genotypic evolution of resistance and its associated fitness costs. I conclude this dissertation with a broader discussion about these and other factors that can influence the evolution of antibiotic resistance, and their clinical and public-health implications.
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